Polypeptide for hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof as well as a polypeptide containing an additive, use of same as well as a process

ABSTRACT

The invention relates to a polypeptide for the hydrolytic cleavage of zearalenone and/or at least one zearalenone derivative, said polypeptide being a hydrolase having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 or a functional variant thereof, wherein the sequence of the functional variant is at least 40% identical to at least one of the amino acid sequences. The invention also relates to: an additive containing the polypeptide; an isolated polynucleotide that encodes the polypeptide; and a method for the hydrolytic cleavage of zearalenone and/or of at least one zearalenone derivative using the polypeptide.

The present invention relates to a polypeptide for hydrolytic cleavageof zearalenone and/or at least one zearalenone derivative, an isolatedpolynucleotide which codes for such a polypeptide as well as an additivecontaining such a polypeptide as well as a use of such a polypeptide aswell as a method for hydrolytic cleavage of zearalenone and/or at leastone zearalenone derivative.

BACKGROUND OF THE INVENTION

Mycotoxins are secondary metabolites produced by filamentary fungi. Animportant representative of mycotoxins is zearalenone (ZEN), which waspreviously known as F-2 toxin, which is produced by a variety ofFusarium fungi and can be found throughout the world. These fungi infestcultivated plants, among others, such as various types of grain, whereinthe fungal infestation usually occurs before the harvest when the growthof the fungi and/or the mycotoxin production may take place beforestorage or may even take place after harvest, either prior to storage orunder improper storage conditions. The FAO has estimated that 25% ofagrarian products throughout the world are contaminated with mycotoxins,thus resulting in substantial economic losses. In an international studyconcluded recently, a total of 23,781 samples were analyzed from January2009 to December 2011, 81% of them testing positive for at least onemycotoxin and 45% testing positive for ZEN. ZEN has been found in allregions of the world and in all types of grain and feed crops tested,such as corn, soy flour, wheat, wheat bran, DDGS (dried distillersgrains with solubles) as well as in finished animal feed mixtures withan incidence of up to 100%.

ZEN is a nonsteroidal estrogenic macrocyclic lactone with the followingstructural formula, synthesized by way of the polyketide metabolicpathway:

and its name according to the IUPAC nomenclature is(2E,11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione.

However, a variety of ZEN derivatives also occurs in nature and may beformed by enzymatic or chemical modifications of ZEN. Examples includeglycosidic ZEN conjugates or those containing sulfate, formed by fungi,plants or a mammalian metabolism as well as ZEN metabolites formed inthe human or animal organism, among others. ZEN derivatives areunderstood below to be ZEN conjugates or ZEN metabolites that occurnaturally or are synthesized by chemical or biochemical synthesis but inparticular α-zearalenol (α-ZEL;(2E,7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]-octadeca-1(18),2,14,16-tetraen-13-one),β-zearalenol (β-ZEL;(2E,7S,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one),α-zearalanol (α-ZAL;(7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octa-deca-1(18),14,16-trien-13-one),β-zearalanol (β-ZAL;(7S,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(14),15,17-trien-13-one), zearalenone 14-sulfate (Z14S;[(2E,11S)-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-17-yl]hydrogen sulfate), zearalenone-14-glycoside (Z14G;(2E,11S)-15-hydroxy-11-methyl-17-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydropyran-2-yl]oxy-12-oxabicyclo[12.4.0]octadeca-1(18)2,14,16-tetraene-7,13-dione)as well as zearalanone (ZAN;(11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo-[12.4.0]octadeca-1(18),14,16-triene-7,13-dione).

ZEN as well as ZEN derivatives, in particular α-ZEL, β-ZEL, Z14S, α-ZAL,β-ZAL, Z14G and ZAN can also be detected in processed foods and animalfeed products, such as bread or beer because of their high chemical andphysical stability.

ZEN binds to the estrogen receptor and can cause hormonal disruptions,being absorbed immediately after oral ingestion and converted by mammalsinto the two stereoisomeric metabolites α-ZEL and/or β-ZEL. For example,α-ZEL but also α-ZAL and/or ZAN have a much stronger estrogenic effectthan ZEN. Meanwhile, conjugated ZEN derivatives have a lower estrogenicactivity than ZEN but ZEN can be released again from these ZENderivatives in the digestive tract under some circumstances.

Although ZEN has a relatively low acute toxicity and has an oral LD₅₀ ofup to 20,000 mg/kg body weight, subacute and/or subchronic toxic effectssuch as teratogenic, carcinogenic, estrogenic and immunosuppressanteffects may occur in animals or humans with prolonged exposure. Feedcontaminated with ZEN leads to developmental disorders in mammaliananimals, but pigs, in particular piglets, are extremely sensitive toZEN. ZEN concentrations of more than 0.5 ppm in feed result indevelopmental disorders, and concentrations of more than 1.5 ppm inpigs, for example, can result in hyperestrogenicity, and concentrationsof 12 ppm ZEN have been blamed for spontaneous abortions in cattle.Since zearalenone is absorbed rapidly through the mucous membranes, inparticular through the gastric mucosa as well as the oral mucosa,immediate and quantitative deactivation is essential. ZEN can bedetected in blood even 30 minutes after oral administration. In thiscase, the use of isolated enzymes offers some advantages with respect tomicroorganisms, such as a higher specific activity or a quicker effect.Because of the harmful effects of ZEN, the European Union has bindingupper limits for ZEN in foodstuffs as well as recommendations for upperlimits for ZEN in animal feed products (EC No. 1881/2006).

The primary strategy for reducing ZEN contamination of foods and animalfeed products is to restrict the growth of fungi, for example, bymaintaining “good agricultural practice.” This includes, among otherthings, ensuring that the seed is free of pests and fungal infestationor that agricultural waste products are removed from the field promptly.In addition, fungal growth in the field can be reduced through the useof fungicides. After the harvest, the harvested material should bestored at a residual moisture level of less than 15% and at a lowtemperature to prevent the growth of fungi. Likewise, materialcontaminated by fungal infestation should be removed before furtherprocessing. Despite the list of measures, I. Rodriges and K. Naehrer(2012) have reported that, even in regions with the highest agriculturalstandards, such as the United States and Central Europe in the years2009 to 2011, 29% and 39% respectively, of the tested corn samples werecontaminated with ZEN.

Additional possibilities for removing ZEN from foodstuffs or animal feedproducts include adsorption and/or transformation of the mycotoxin. Thisrequires that binding of the mycotoxin to the adsorbent must be strongand specific over a wide pH range and must remain stable in thegastrointestinal tract. Although some nonbiological adsorbents such asactivated carbon and silicates or synthetic polymers such ascholestyramine can be used efficiently for aflatoxins, their use forother mycotoxins is limited. The main disadvantage of adsorbents is thenonspecific binding of other molecules, which are in some casesessential for nutrition. Biological adsorbents such as yeast or yeastextracts have also been described in the literature but have alimitation similar to that of nonbiological adsorbents.

Detoxification of ZEN by physical and chemical treatments is alsolimited. ZEN cannot be deactivated effectively by thermal treatment, butthe ZEN content can be reduced by 83.9% by extrusion and treatment withoxidizing agents, for example, for 16 hours at 80° C. with 10% hydrogenperoxide solution. Use of extrusion methods and oxidizing agents such asozone or hydrogen peroxide in the production of foodstuffs and animalfeed products is limited because of the high cost, the loss of qualityand in some cases the low efficacy and low specificity.

Biotransformation of ZEN by means of microorganisms such as Trichosporonmycotoxinivorans, Gliocladium roseum or Bacillus subtilis strains and/orenzymes isolated from them such as hydrolases or peroxidases hisdescribed, for example, by E. Vekiru et al. in Appl. and Environ.Microb., 2010, 76, 7, 2353-2359.

EP 0 938 575 B1 has described ZEN-degrading properties of bacteria ofthe genus Rhodococcus or Nocardia, in particular R. globerulus, R.erythropolis and N. globerula.

WO 02/076205 describes the ZEN-degrading effect of enzymes isolated fromGliocladium roseum, including α,β-hydrolase and zearalenone hydrolase 1(ZHD1), which catalyze the degradation of ZEN by means of a catalytictriad.

WO 2012/113827 discloses recombinant zonases, namely enzymes thatdegrade ZEN and remain stable in the gastrointestinal tract. Theseinclude microorganisms such as Thermobifida fusca, Streptomycesexfoliates, Acidovorans delafieldii and Streptomyces sp. in particular.

Polypeptides or enzymes capable of hydrolyzing ZEN and/or at least oneZEN derivative may also be designated as zonases.

The terms used hereinafter are taken from the technical language andeach is used in the traditional meanings, unless something to thecontrary is indicated. Thus, for example, the term “polynucleotide”relates to all types of genetic material of all lengths and sequencessuch as single-stranded and double-stranded DNA and RNA molecules,including regulatory elements, structural elements, groups of genes,plasmids, entire genomes and fragments thereof. The designation“polypeptide” Includes proteins such as, for example, enzymes,antibodies as well as polypeptides with up to 500 amino acids, such as,for example, peptide inhibitors, domains of proteins or also shortpolypeptides with short sequence lengths, for example, less than 10amino acids, such as receptors, ligands, peptide hormones, tags and thelike. The designation “position” in a polynucleotide or polypeptiderelates to a single specific base or amino acid in the sequence of thepolynucleotide or of the polypeptide.

BRIEF SUMMARY OF THE INVENTION

The present invention is now aimed at making available a polypeptidewith which it is possible to rapidly and reliably transform ZEN and/orat least one ZEN derivative into hydrolyzed ZEN and/or hydrolyzed ZENderivatives.

To achieve this object, the present invention is characterizedessentially in that the polypeptide is a hydrolase with an amino acidsequence selected from the group of sequence ID numbers 1 to 15 or afunctional variation thereof, wherein there is a sequence identity of atleast 70% between the functional variant and at least one of the aminoacid sequences.

The term “sequence identity” according to the present invention relatesto a percentage sequence identity. For amino acid sequences andnucleotide sequences, the sequence identity can be determined visually,but is preferably calculated by a computer program. The sequencecomparison is also carried out within sequence segments, wherein thesegment is understood to be a continuous sequence of the referencesequence and preferably comprises a conserved region of the sequence.

In the present case, the sequence identity was determined with the helpof the NCBI BLAST program (BLAST=Basic Logic Alignment Search Tool), inparticular with BLASTP for polypeptides and BLASTN for polynucleotides,which are made available on the homepage of the National Center forBiotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). It isthus possible to compare two or more sequences with one anotheraccording to the algorithm of Altschul et al., 1997 (Nucleic Acids Res.,25:3389-3402). For this purpose of this invention, the programs wereused in the version of 15 May 2013. The basic settings were used as theprogram settings, but in particular for the amino acid sequencecomparison: “max target sequence”=100; “expected threshold”=10; “wordsize”=3; “matrix”=BLOSOM62; “gap costs”=“existence: 11; extension: 1”;“computational adjustment”=“conditional compositional score matrixadjustment” as well as for the nucleotide sequence comparison word size:11; expect value: 10; gap costs: existence=5, extension=2; filter=lowcomplexity activated; match/mismatch scores: 2-3; filter string: L; m.

The terms “functional polypeptide variant” or “functional variant”relate first to “allelic variants” of the polypeptide and to “functionalfragments” of the polypeptide and secondly to “modification” of thepolypeptide, wherein the enzymatic function is essentially unchanged.The term “allelic variant” relates to a polypeptide formed by naturallyoccurring mutation(s) in the nucleotide sequence and causing a change inthe amino acid sequence, wherein the enzymatic function thereof is notaffected. “Modifications” may be, for example, C- or N-terminal fusionswith polypeptides or mutated polypeptides, wherein mutations can beobtained by substitution, insertion or deletion of at least one aminoacid, in particular by site-directed mutagenesis, i.e., randommutagenesis, recombination and/or any other protein engineering method.The terms “substitution,” “insertion” and “deletion” are used here inthe common meanings in genetic engineering, with which those skilled inthe art are familiar. The term “functional fragment” refers to a part ora subsequence of a polypeptide or a part and/or a subsequence of afunctional variant thereof, wherein the enzymatic function isessentially retained. An enzymatic function is retained in particularwhen the enzymatic reaction mechanism remains unchanged, i.e., themycotoxin is hydrolyzed in the same location, and the specific residualactivity “functional variant” amounts to at least 5%, preferably atleast 10%, especially at least 10%, and in particular at least 50%,based on the original polypeptide. The polypeptides with the amino acidsequences having the sequence ID numbers 1 through 15 are functionalallelic variants either of one another or of one and the same enzyme,wherein the sequences originate from different microorganisms. This isclearly recognizable from the close relationship to one another,measured by means of the percentage sequence identity, as well as thefact that all polypeptides act on ZEN and ZEN derivatives by means ofthe same degradation mechanisms.

Because of the similarity in the amino acid sequences of thepolypeptides with the sequence ID numbers 1 through 15 to one another,it is possible that a functional variant of one of these polypeptidesmay have a sequence identity of at least 40%, with more than one of theclaimed polypeptides having the sequence ID numbers 1 through 15.

Through the choice of such an amino acid sequence or a functionalvariant thereof, a surprisingly fast and complete hydrolysis of ZENand/or at least one ZEN derivative has been detected.

As corresponds to a preferred further development of the invention, thepolypeptide has an amino acid sequence, which contains at least oneconserved amino acid sequence segment or a functional variant thereof,wherein the functional variant of the amino acid sequence segment has asequence identity of at least 70%, preferably at least 84%, morepreferably at least 92% and most preferably at least 98%, and the atleast one conserved amino acid sequence segment is selected from thegroup of amino acid sequences +24 to +50, +52 to +77, +79 to +87, +89 to+145, +150 to +171, +177 to +193, +223 to +228, +230 to +237, +239 to+247, +249 to +255, +257 to +261, +263 to +270, +272 to +279, +297 to+301, +303 to +313, +24 to 328, +1 to +328 of the sequence having thesequence ID no. 1. Due to the presence of at least one such conservedamino acid sequence segment, it has been possible to make available apolypeptide which also has, in addition to the rapid and completehydrolysis of ZEN and/or of at least one ZEN derivative, a particularlyhigh activity value in comparison with ZEN degrading polypeptides knownpreviously.

Equally good results have been achieved when the functional variant hasat least one amino acid modification selected from the group ofsubstitution, deletion and insertion of one or more amino acids.

If the polypeptide has a specific activity of at least 0.01 U/mg,preferably at least 0.1 U/mg, in particular at least 1 U/mg; and/or aK_(M) value of the hydrolytic cleavage of ZEN of at most 50 μM,preferably at most 3.5 μM, in particular at most 0.5 μM; and/or ak_(cat) value of the hydrolytic cleavage of ZEN of at least 0.05 s⁻¹,preferably at least 0.6 s⁻¹, in particular at least 5 s⁻¹; and/or av_(max) value of the hydrolytic cleavage of ZEN of at least 0.00001 μM⁻¹s⁻¹, preferably at least 0.0001 μM⁻¹ s⁻¹, in particular at least 0.001μM⁻¹ s⁻¹, then ZEN and/or ZEN derivatives can be hydrolyzed especiallyrapidly and completely, in particular being detoxified.

In addition, the polypeptide may contain an amino acid sequence selectedfrom the group of sequence ID numbers 5, 6 and 15 or a functionalvariant thereof, wherein the functional variant has at least 70%sequence identity with at least one of the amino acid sequences, and thepH stability of the polypeptide at pH 5.0 amounts to at least 15%,preferably 50% and in particular preferably 90%. It is possible in thisway to ensure that the polypeptide zearalenone and/or at least onezearalenone derivative will be cleaved and/or detoxified even in anacidic medium, such as the mammalian stomach, for example. The pHstability of polypeptides is defined here as the percentage residualactivity of the polypeptides at pH 5.0 in relation to the activity atthe respective optimum pH.

In addition, the polypeptide may contain an amino acid sequence selectedfrom the group of sequence ID numbers 1, 5, 6 and 15 or a functionalvariant thereof, wherein the functional variant has at least 70%sequence identity with at least one of the amino acid sequences, so thatthe polypeptide has the highest enzymatic activity in a temperaturerange between 30° C. and 75° C., preferably between 38° C. and 55° C.,in particular preferably between 38° C. and 52° C. This ensures thatzearalenone and/or at least one zearalenone derivative is alsohydrolyzed and/or detoxified by the polypeptide even at mesophilictemperatures, in particular at the body temperature of humans and farmanimals. The temperature at which the polypeptide has the highestenzymatic activity is defined as the optimum temperature of thepolypeptide.

Finally, the polypeptide may have an amino acid sequence selected fromthe group of sequence ID numbers 1, 5, 6 and 15 or a functional variantthereof, wherein the functional variant has at least 70% sequenceidentity with at least one of the amino acid sequences, and thepolypeptide is thermally stable up to a temperature of 90° C.,preferably 75° C. and in particular preferably 60° C. This ensures thatthe polypeptide and its enzymatic function will remain essentiallyintact even under elevated temperature stress, such as that which mayoccur, for example, during shipping in a container or duringpelletization of feed. The thermal stability of polypeptides is definedas the temperature at which, after 15 minutes of preliminary incubation,the polypeptide has a 50% residual activity in comparison with theactivity at the respective optimum temperature.

The polypeptide may be selected so that it has an α,β-hydrolase, whichis suitable for oxygen-independent and cofactor-free hydrolytic cleavageof the ester group of zearalenone and/or of the ZEN derivatives, whichhas an amino acid triad that catalyzes the hydrolytic cleavage andconsists of serine, an acidic amino acid selected from glutamic acid andaspartic acid, in particular aspartic acid and histidine, and thecatalytic triad is, for example, S128, D264 and H303, wherein thepositioning relative to the sequence ID no. 1 is shown.

Hydrolysis of ZEN and ZEN derivatives succeeds with any of thepolypeptides of the sequence ID numbers 1 to 15 on the ester group ofzearalenone or its derivatives according to the following reactionmechanism:

The hydrolysis of ZEN to form nontoxic hydrolyzed zearalenone (HZEN)and/or hydrolyzed ZEN derivatives takes place by means of polypeptidesaccording to the invention, in particular α,β-hydrolases. The furtherdecarboxylation of HZEN to decarboxylated hydrolyzed ZEN (DHZEN) and/ordecarboxylated hydrolyzed ZEN derivatives is usually spontaneous.

In particular, by means of the aforementioned catalytic triads, it ispossible to completely hydrolyze ZEN and ZEN derivatives, wherein thedegradation reaction has a good pH stabilizing effect, in particular ata pH in the acidic range.

It has been found that it is possible to achieve uniformly good resultswith a polypeptide that contains in a sequence segment consisting ofthree amino acids before the serine and three amino acids after theserine of the aforementioned catalytic triad, at least one polar aminoacid selected from Y, Q, N, T, K, R, E, D and at least one nonpolaramino acid selected from F, M, L, I, V, A, G, P, and it is also possibleto improve at least one enzyme kinetic parameter.

In a preferred refinement of the invention, the polypeptide has at leastone mutation of the amino acid sequence with respect to the sequence IDno. 1 in at least one of the following positions: 22, 23, 25, 26, 27,29, 31, 32, 35, 37, 42, 43, 46, 51, 53, 54, 57, 60, 69, 72, 73, 78, 80,84, 88, 95, 97, 99, 114, 118, 119, 123, 132, 141, 146, 148, 149, 154,163, 164, 165, 169, 170, 172, 176, 180, 182, 183, 190, 191, 194, 196,197, 198, 201, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 216,217, 220, 221, 222, 229, 231, 233, 238, 240, 244, 245, 246, 248, 249,251, 254, 256, 260, 262, 263, 266, 269, 271, 277, 280, 281, 282, 283,284, 285, 286, 287, 292, 296, 298, 302, 307, 308, 309, 311, 314, 317,319, 321, 323, 325 and 326. These positions are derived from thesequence differences between the polypeptide with the sequence ID no. 1and the polypeptides having the sequence ID numbers 2 to 6, which areespecially active and have a high degree of identity with this sequence.If the polypeptide with sequence ID no. 1 is modified in at least one ofthese positions, so that the amino acid variants of sequence ID numbers2 through 6 can be taken over in this position, it is possible to showthat these positions have a significant influence on the enzyme kineticparameters of the polypeptide and that combinations of the sequence IDno. 1 with sequence ID numbers 2 through 6 also having a high degree ofsequence identity in addition will lead to higher activities.

According to one refinement of the invention, the polypeptide has atleast one mutation selected from the group comprising: D22A, S23Q, S23L,N25D, I26V, F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V37A, V42I, V43T,F46Y, S51E, S51D, D53G, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S,N80H, F84Y, I88L, T95S, T97A, R99K, I114M, I118V, K119R, V123I, L132V,A141 S, I146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H,V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, G191S,K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S,P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, ΔP212, T213V,P214A, E216T, E216G, A217I, N220H, L221M, K222R, K222Q, G229A, A231V,F233W, F233Y, F233H, A238G, H240N, H240S, D244E, R245Q, M246L, S248T,8248N, S248G, Q249R, K251N, I254V, I256L, A260M, T262D, T262G, I263T,E266D, E269H, E269N, L271V, L277E, E280A, E280L, H281R, H281Q, A282V,Q283R, D284L, D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A,Q296E, H298V, L302S, L307Q, F308S, D309A, A311P, A314V, L317F, S319Q,S319P, S319R, S321A, S321T, T323A, P325A, A326P in the amino acidsequence with respect to sequence ID no. 1. With such a polypeptide, itis possible to completely hydrolyze ZEN within a short period of time,in particular to detoxify it, wherein the specific activity of thepolypeptide amounts to at least 6.00 U/mg, preferably at least 7.00U/mg, in particular at least 8.00 U/mg. The unit “U” or also “unit” is ameasure of the absolute catalytic activity and is defined by thehydrolysis of 1 μmol ZEN per minute at 32° C. in 50 mM Tris-HCl buffer(pH 8.2), wherein “catalytic activity” is understood to refer to theenzymatic conversion of a substrate under defined reaction conditions,and “specific activity” is understood to refer to the ratio of thecatalytic activity and the polypeptide mass concentration (mass per unitof volume).

If, according to one refinement of the invention, the polypeptide isembodied so that at least one of the following amino acid motifs with asequence having sequence ID numbers 32 to 50 is contained in it, it isthen possible to make available polypeptides having a specific activityof at least 17.00 U/mg, preferably at least 8.00 U/mg. It hassurprisingly been found that when at least one of the following aminoacid motifs with a sequence having the sequence ID numbers 51 to 58 iscontained in it, the enzymatic activity of the polypeptide is increasedfurther, for example, in comparison with a motif containing seven aminoacids. An even higher specific activity is achieved when at least one ofthe following amino acid motifs having the sequence having the sequenceID numbers 59 to 69 is contained in it.

Finally, the polypeptide may contain at least one conservative aminoacid substitution in at least one position, where the conservative aminoacid substitution is selected from substitutions of G to A; or A to G,S; or V to I, L, A, T, S; or I to V, L, M; or L to I, M, V; or M to L,I, V; or P to A, S, N; or F to Y, W, H; or Y to F, W, H; or W to Y, F,H; or R to K, E, D; or K to R, E, D; or H to Q, N, S; or D to N, E, K,R, Q; or E to Q, D, K, R, N; or S to T, A; or T to S, V. A; or C to S,T, A; or N to D, Q, H, S; or Q to E, N, H, K, R, wherein the designation“conservative amino acid substitution” relates to the substitution ofamino acids by other amino acids regarded by those skilled in the art asbeing conservative, i.e., having similar specific properties. Suchspecific properties include, for example, the size, polarity,hydrophobicity, charge or pKs value of the amino acid. A conservativemutation, for example, is understood to be a substitution of one acidicamino acid for another acidic amino acid, a basic amino acid for anotherbasic amino acid or a polar amino acid for another polar amino acid.

With such conservative amino acid substitutions, it is possible toproduce functional polypeptide variants whose specific activity isapproximately the same in comparison with the parental polypeptide butis preferably increased by at least 0.1 U/mg.

The present invention is additionally aimed at making available anisolated polynucleotide with which it is possible to synthesize apolypeptide for rapid and reliable hydrolytic cleavage of ZEN and/or ofat least one ZEN derivative.

To achieve this object, the invention is characterized in that theisolated polynucleotide may have a nucleotide sequence that codes for apolypeptide, wherein the polypeptide has a zearalenone and/or theproperty of hydrolyzing at least one zearalenone derivative, and thenucleotide sequence codes for at least one polypeptide according to theinvention and/or the nucleotide sequence has a degree of sequenceidentity of at least 70% with a nucleotide sequence selected from thegroup of sequence ID numbers 16 to 31 and/or the nucleotide sequencehydrolyzes under moderate stringency conditions with at least onenucleotide sequence selected from the group of sequence ID numbers 16 to31 and/or with a subsequence thereof with at least 200 nucleotides, inparticular at least 100 nucleotides and/or with a complementary strandof the nucleotide sequence or subsequences thereof.

Nucleotide sequences to be expressed, in particular their triplets(codons) are usually altered depending on the host cell so that thecodon bias is optimized according to the host cell. This results in thefact that even polynucleotides having a degree of sequence identity offar less than 80% but even less than 70% or less than 60% can code forthe same polypeptide. The sequence comparison for determining the degreeof sequence identity must also be performed within sequence segments,wherein one section is to be understood as a continuous sequence of thereference sequence. The length of the sequence segments for nucleotidesequences is normally 15 to 600.

With the help of the present isolated nucleotide sequences or sequencesegments, it is possible to generate nucleic acid probes having a lengthof usually at least 15, 30 or 40 nucleotides. With such probes, whichare typically also labeled, e.g., by ³H, ³²P, ³⁵S, biotin or avidine, itis possible, by using standard methods, to identify nucleotide sequencesthat code for polypeptides with the property of degrading ZEN and/or ZENderivatives. For example, DNA, RNA or cDNA from individualmicroorganisms, genomic DNA libraries or cDNA libraries can be used asthe starting material for identification of such sequences.

For nucleotide sequences and/or nucleotide probes with a length of atleast 100 nucleotides, moderate stringency conditions are defined asprehybridization and hybridization at 42° C. in Na-EDTA buffer providedwith 5× NaCl (SPE, 0.9M NaCl, 60 mM NaH₂PO₄, 6 mM EDTA) containing 0.3%sodium dodecyl sulfate (SDS), 200 μg/mL sheared and denatured salmonsperm DNA and 35% formamide followed by standard Southern Blotconditions, wherein the carrier material is washed three times at theend for 15 minutes each with 2× sodium chloride citrate buffer (SSC, 300mM NaCl and 30 mM trisodium citrate, 0.2% SDS) at 55° C.

For nucleotide sequences and/or nucleotide probes with a length of 15nucleotides to 100 nucleotides, moderate stringency conditions aredefined as prehybridization and hybridization in buffer consisting of0.9M NaCl, 0.09M Tris-HCl pH=7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate,0.1 mM ATP and 0.2 mg/mL yeast RNA, wherein prehybridization andhybridization are performed at a temperature 5° C. to 10° C. below thecalculated melting point (Tm), where Tm is determined by calculationaccording to Bolton and McCarthy (1962, Proceedings of the NationalAcademy of Sciences USA, 48:1390). Following this, the experiment iscontinued under standard Southern Blot conditions (J. Sambrook, E. F.Fritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2^(nd) edition, Cold Spring Harbor, N.Y.). The carrier material iswashed at the end once for 15 minutes with 6×SCC buffer [sic; SSCbuffer] containing 0.1% SDS and twice for 15 minutes with 6×SSC buffereach at 5° C. to 10° C. under the calculated Tm.

The present invention is additionally aimed at making available anadditive with which it is possible to achieve a rapid and reliablehydrolytic cleavage of ZEN and/or at least one ZEN derivative in adefined or complex matrix, such as, for example, in food or animal feedproducts.

To achieve this goal, an additive that hydrolytically cleaves azearalenone and/or at least zearalenone derivative is made available foranimal feed products for pigs, poultry or aquaculture, for foodstuffs orDDGS (distillers dried grain and solubles), wherein the additivecontains at least one polypeptide having an amino acid sequence selectedfrom the group of sequence ID numbers 1 to 15 or a functional variantthereof, wherein the sequence identity between the functional variantand at least one of the amino acid sequences amounts to at least 70%,and auxiliary substances are also present.

With such an additive, the biochemical conversion of ZEN and/or at leastone ZEN derivative to hydrolyzed ZEN and/or hydrolyzed ZEN derivative ispossible. This additive can also be used, for example, forstereoselective hydrolysis of ZEN and/or ZEN derivatives in industrialprocesses.

In a preferred refinement of the invention, the additive is embodied sothat the auxiliary substances are selected from at least one inertcarrier as well as optionally additional ingredients, such as vitaminsand/or minerals and/or enzymes and/or additional components fordetoxification of mycotoxins. Due to the use of such an additive, forexample, in foodstuffs or animal feed products, it is possible to ensurethat any amounts of ZEN and/or ZEN derivatives that might be present arereliably hydrolyzed, in particular being detoxified to the extent thatthey will have no harmful effect on the organism of the subjectconsuming this foodstuff or animal feed product.

A polypeptide according to the invention here may also be present in anenzyme preparation, which additionally contains at least one enzyme inaddition to at least one polypeptide according to the invention, suchthat the enzyme takes part in the degradation of proteins, for example,such as proteases, or plays a role in the metabolism of starch or fiberor fat or glycogen, such as, for example, amylase, cellulase orglucanases as well as, for example, hydrolases, lipolytic enzymes,mannosidase, oxidases, oxidoreductases, phytases, xylanases and/orcombinations thereof.

Additional fields of use of the invention include enzyme preparations,which, in addition to at least one polypeptide according to theinvention, also contain at least one component for detoxification ofmycotoxins, such as a mycotoxin-degrading enzyme, for example, aflatoxinoxidase, ergotamine hydrolases, ergotamine amidases, zearalenoneesterases, zearalenone lactonases, ochratoxin amidases, fumonisincarboxyl esterases, fumonisin aminotransferases, aminopolyolaminooxidases, deoxynlvalenol epoxide hydrolases and/or at least onemycotoxin-degrading microorganism, such as Bacillus subtilis and/or atleast one mycotoxin-binding component, for example, microbial cell wallsor inorganic materials such as bentonites.

According to one particularly preferred refinement of the invention, atleast one polypeptide is present in the additive in a concentration ofat most 10,000 U/g, preferably at most 1000 U/g, more preferably at most100 U/g and most preferably at most 10 U/g, so that it is possible toconvert ZEN and/or ZEN derivatives rapidly and in particular to do soalready before they are absorbed by the body of a subject, in particulara mammal consuming a contaminated foodstuff or animal feed product,converting them into nontoxic or less toxic metabolites, in particularHZEN and DHZEN.

According to a refinement of the invention, the polypeptide is presentin encapsulated or coated form, wherein standard methods such as thosedescribed in WO 92/12645 can be used for the encapsulation or coating.By encapsulation and/or coating, it is possible to transport thepolypeptide without any change, in particular without degradation ordamage, to its site of use, so that only after the protective shell hasbeen dissolved in the digestive tract of animals, for example, does thepolypeptide begin to act so that an even more targeted, rapid andcomplete degradation of ZEN and/or ZEN derivatives can be achieved evenin the acidic protease-rich and anaerobic medium. In addition, it isalso possible through encapsulation or coating to increase the thermalstability of the polypeptides in the additive.

The present invention is additionally aimed at use of an additivecontaining at least one polypeptide having an amino acid sequenceselected from the group of sequence ID numbers 1 to 15 or a functionalvariant thereof, wherein the sequence identity between the functionalvariant and at least one of the amino acid sequences amounts to at least70% for hydrolytic cleavage of zearalenone and/or at least onezearalenone derivative in animal feed products, in particular for pigs,poultry and agriculture, in foodstuffs or in distillers dried grain andsolubles. Through the use of the additive according to the Invention, itis possible to hydrolyze and/or detoxify the ZEN and/or ZEN derivativescontained in the foodstuff or animal feed product and/or distillersdried grain and solubles, wherein such a detoxification is possible evenwith polypeptide concentrations of approximately 1 U/g contaminatedfoodstuff or animal feed product.

The present invention is additionally aimed at making available a methodwith which a rapid and reliable hydrolytic cleavage of ZEN and/or atleast one ZEN derivative is made possible.

To achieve this goal, this method is carried out in such a way thatzearalenone and/or at least one zearalenone derivative having an aminoacid sequence selected from the group of sequence ID numbers 1 to 15 ora functional variant thereof is hydrolyzed, wherein the sequenceidentity between the functional variant and at least one of the aminoacid sequences amounts to at least 70%.

According to one refinement of the invention the method is carried outin such a way that the polypeptide therein is used in an additivecorresponding to this invention.

According to another preferred refinement, the method is carried out insuch a way that the polypeptide or the additive therein is mixed with afoodstuff or animal feed product contaminated with zearalenone and/orwith at least one zearalenone derivative; the contaminated foodstuff oranimal feed product is brought in contact with moisture and thepolypeptide or the additive hydrolyzes the zearalenone and/or at leastone zearalenone derivative contained in the contaminated foodstuff oranimal feed product. In the case of moist foodstuffs or animal feedproducts, such as mash or slurries, the hydrolysis of the zearalenoneand/or of at least one zearalenone derivative will take place in themoist foodstuff or animal feed product before oral consumption. Due tothis method, it is possible to ensure that the harmful effects ofzearalenone and zearalenone derivatives on humans and animals will belargely eliminated. Moisture here is understood to refer to the presenceof water or aqueous liquids, which also include, for example, saliva orother liquids present in the digestive tract. The digestive tract isdefined as the oral cavity, the pharynx (throat), the esophagus and thegastrointestinal tract or equivalents thereof, wherein there may bedifferent designations for animals and/or individual components may notoccur in the digestive tract of animals.

The method according to the invention may also be carried out in such away that the foodstuff or animal feed product is pelletized before oralconsumption.

According to one refinement of the invention, the method is carried outso that at least 70%, preferably at least 80% in particular at least 90%of the zearalenone and/or at least one zearalenone derivative ishydrolyzed. Therefore, subacute and/or chronic toxic effects such asteratogenic, carcinogenic, estrogenic and immunosuppressant effects inanimals or humans, for example, can be suppressed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The invention is explained in greater detail below on the basis ofexemplary embodiments as well as drawings, in which:

FIG. 1 shows the degradation of ZEN and the increase in the metabolitesHZEN and DHZEN over time for the polypeptide having the sequence ID no.1, wherein the polypeptide in FIG. 1A has not been tagged, in FIG. 1Bthe polypeptide has a C-terminal 6×His tag, and in FIG. 1C thepolypeptide has an N-terminal 6×His tag.

FIG. 2 shows the Michaelis-Menten kinetics of the polypeptide withsequence ID no. 1, FIG. 2A shows the Michaelis-Menten kinetic of thefirst measurement (according to table 3) and FIG. 2B shows theMichaelis-Menten kinetic of the second measurement (according to table3).

FIG. 3 shows the degradation of ZEN and the increase in metabolites HZENand DHZEN over time, due to purified polypeptides having the sequence IDnumbers 1 (FIG. 3A), 2 (FIG. 3B), 5 (FIG. 3C), 6 (FIG. 3D), 7 (FIG. 3E),9 (FIG. 3F), 11 (FIG. 3G), 12 (FIG. 3H) and 15 (FIG. 3I), wherein allthe sequences have a C-terminal 6×His tag.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Modification, Cloningand Expression of Polynucleotides that Code for Polypeptides which areCapable of Hydrolytic Cleavage of ZEN and/or at Least One ZEN Derivative

Amino acid substitutions, insertions or deletions were performed bymutation of the nucleotide sequences by means of PCR using the “quickchange site-directed mutagenesis kits” (Stratagene) according to theinstructions. As an alternative, complete nucleotide sequences were alsoordered (GeneArt). The nucleotide sequences generated by means of PCRmutagenesis and/or ordered from GeneArt optionally also contained a C-or N-terminal 6×His tag on an amino acid level and were integrated bymeans of standard methods into expression vectors for expression in E.coli or P. pastoris, transformed in E. coli or P. pastoris and expressedin E. coli and P. pastoris (J. M. Cregg, Pichia Protocols, secondedition, ISBN-10: 1588294293, 2007; J. Sambrook et al., 2012, MolecularCloning, A Laboratory Manual, 4^(th) edition, Cold Spring Harbor),wherein any other suitable host cell may also be used for this task.

The designation “expression vector” relates to a DNA construct that iscapable of expressing a gene in vivo or in vitro. In particular this isunderstood to refer to DNA constructs that are suitable for transferringthe polypeptide coding nucleotide sequence into the host cell tointegrate into the genome there or to be present freely in theextrachromosomal space and to express the polypeptide coding nucleotidesequence intracellularly and optionally also to remove the polypeptidefrom the cell.

The designation “host cell” refers to all cells containing either anucleotide sequence to be expressed or an expression vector and beingcapable of synthesizing a polypeptide according to the invention. Inparticular this is understood to include prokaryotic and/or eukaryoticcells, preferably P. pastoris, E. coli, Bacillus subtilis, Streptomyces,Hansenula, Trichoderma, Lactobacillus, Aspergillus, plant cells and/orspores of Bacillus, Trichoderma or Aspergillus.

The soluble cell lysate in the case of E. coli and/or the culturesupernatant in the case of P. pastoris was/were used for determinationof the catalytic properties of the polypeptides. To determine the K_(M)value, v_(max), k_(cat) and the specific activity, the polypeptides wereselectively enriched chromatographically by standard methods overnickel-Sepharose columns. The determination of the protein concentrationwas performed by means of standard methods, either being calculated bythe BCA method (Pierce BCA Protein Assay KitProd #23225) or preferablyphotometrically with the specific extinction coefficients for therespective proteins that are available online with the ProtParam programat http://web.expasy.org/protparam (Gasteiger E. et al.; ProteinIdentification and Analysis Tools on the ExPASy Server, in John M.Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005, pp.571-607).

Example 2 Determination of the Sequence Identity and the Conserved AminoAcid Sequence Segments

The determination of the percentage sequence identity based on the totalpolypeptide length of the polypeptides with eh amino acid sequenceshaving the sequence ID numbers 1 to 15 relative to one another (Table 1)was performed with the help of the BLAST program (Basic Local AlignmentSearch Tool), in particular with BLASTP, which can be used at homepageof the National Center for Biotechnology Information (NCBI;http://www.ncbi.nlm.nih.gov/). It is thus possible to compare two ormore sequences with one another according to the algorithm of Altschulet al., 1997 (Nucleic Acids Res. (1997), 25:3389-3402). The basicsettings were used as the program settings in particular. However. “maxtarget sequence”=100; expected threshold”=10; “word size”=3;“matrix”=BLOSOM62; “gap costs”=“existence: 11; extension: 1”;“computational adjustment”=“conditional compositional score matrixadjustment.”

To determine the conserved amino acid sequence segments, thepolypeptides having sequence ID numbers 1 to 6, which have a sequenceidentity of at least 70% with one another, were compared with the helpof the COBALT software (J. S. Papadopoulos and R. Agarwala, 2007,COBALT: Constraint-Based Alignment Tool for Multiple Protein Sequences,Bioinformatics 23:1073-79) while using the standard parameters, inparticular the parameters (“gap penalties”: −11, −1; “end-gappenalties”: −5, −1; “use RPS BLAST”: on; “Blast E-value”: 0.003; “findconserved columns and recompute”: on; “use query clusters”: on; “wordsize”: 4; “may cluster distance”: 0,8; “alphabet”: regular; “homologyconversation setting”: 3 bits). The result of this analysis representsthe conserved amino acids. The following ranges of at least fivesuccessive conserved amino acids were defined as the conserved aminoacid sequence segments, namely with respect to the segment having thesequence ID no. 1, the segments A from position +24 to position +50, Bfrom position +52 to position +77, C from position +79 to position +87,D from position +89 to position +145, E from position +150 to position+171, F from position +177 to position +193, G from position +223 toposition +228, H from position +230 to position +237, I from position+239 to position +247, J from position +249 to position +255, K fromposition +257 to position +261, L from position +263 to position +270, Mfrom position +272 to position +279, N from position +297 to position+301 and O from position +303 to position +313.

The determinations of the percentage sequence identity of thepolypeptides to one another and of the conserved amino acid sequencesegments of the individual polypeptides relative to the conserved aminoacid sequence segments of the sequence having the sequence ID no. 1 wereformed as described above. The results are presented in Tables 1 and 2.

TABLE 1 Percentage sequence identity of the polypeptides to one another.SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 1 No. 2 No. 3 No. 4No. 5 No. 6 No. 7 SEQ ID — 70% 71% 71% 71% 71% 64% No. 1 SEQ ID 70% —81% 83% 81% 83% 63% No. 2 SEQ ID 71% 81% — 95% 99% 92% 60% No. 3 SEQ ID71% 83% 95% 95% 95% 60% No. 4 SEQ ID 71% 81% 99% 95% — 93% 60% No. 5 SEQID 71% 83% 92% 95% 93% — 61% No. 6 SEQ ID 64% 63% 60% 60% 60% 61% — No.7 SEQ ID 57% 54% 54% 53% 53% 53% 53% No. 8 SEQ ID 50% 50% 53% 53% 53%55% 51% No. 9 SEQ ID 55% 52% 55% 54% 55% 53% 52% No. 10 SEQ ID 53% 51%53% 51% 51% 52% 54% No. 11 SEQ ID 50% 49% 50% 50% 50% 49% 51% No. 12 SEQID 55% 49% 51% 51% 51% 52% 54% No. 13 SEQ ID 73% 65% 69% 70% 69% 68% 80%No. 14 SEQ ID 79% 68% 71% 71% 71% 72% 63% No. 15 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 8 No. 9 No. 10 No. 11 No. 12 No.13 No. 14 No. 15 SEQ ID 57% 50% 55% 53% 50% 55% 73% 79% No. 1 SEQ ID 54%50% 52% 51% 49% 49% 65% 68% No. 2 SEQ ID 54% 53% 55% 53% 50% 51% 69% 71%No. 3 SEQ ID 53% 53% 54% 51% 50% 51% 70% 71% No. 4 SEQ ID 53% 53% 55%51% 50% 51% 69% 71% No. 5 SEQ ID 53% 55% 53% 52% 49% 52% 68% 72% No. 6SEQ ID 53% 51% 52% 54% 51% 54% 80% 63% No. 7 SEQ ID — 50% 49% 51% 49%48% 83% 51% No. 8 SEQ ID 50% — 51% 52% 69% 51% 67% 51% No. 9 SEQ ID 49%51% — 76% 52% 52% 63% 56% No. 10 SEQ ID 41% 50% 76% — 52% 51% 58% 52%No. 11 SEQ ID 49% 52% 52% 52% — 49% 71% 51% No. 12 SEQ ID 48% 51% 52%51% 49% — 54% 53% No. 13 SEQ ID 83% 67% 63% 58% 71% 55% — 72% No. 14 SEQID 51% 51% 56% 52% 51% 53% 72% — No. 15

TABLE 2 Percentage sequence identity of the conserved amino acidsequence segments A to O. Sequence identity relative to the sequence IDno. 1 Segment Segment Segment Segment Segment Segment Polypeptide A B CD E F SEQ ID No. 1  100%  100%  100%  100%  100%  100% SEQ ID No. 259.6% 76.9% 88.9% 87.7% 77.3% 76.5% SEQ ID No. 3 63.0% 76.9% 77.8% 89.5%86.4% 76.5% SEQ ID No. 4 63.0% 80.8% 77.8% 91.2% 86.4% 76.5% SEQ ID No.5 63.0% 76.9% 77.8% 87.7% 86.4% 76.5% SEQ ID No. 6 63.0% 80.8% 77.8%91.2% 86.4% 76.5% SEQ ID No. 7 44.7% 69.2% 77.8% 78.9% 68.2% 64.7% SEQID No. 8 40.7% 50.0% 66.7% 82.5% 59.1% 64.7% SEQ ID No. 9 51.9% 57.7%55.6% 73.7% 45.5% 58.8% SEQ ID No. 10 44.4% 61.5% 77.8% 75.4% 47.8%76.5% SEQ ID No. 11 44.4% 50.0% 66.7% 71.9% 43.5% 58.8% SEQ ID No. 1251.9% 53.8% 55.6% 71.9% 50.0% 58.8% SEQ ID No. 13 18.5% 61.5% 55.6%77.2% 54.5% 52.9% SEQ ID No. 14 55.6% 69.2% 77.8% 84.2% 54.5% 52.9% SEQID No. 15 74.1% 86.7% 88.9% 89.0% 77.3% 88.2% Sequence identity relativeto the SEQ ID No. 1 Segment Segment Segment Segment Segment SegmentPolypeptide G H I J K L SEQ ID No. 1 100%  100%  100%  100%  100%  100%SEQ ID No. 2 100% 87.5% 66.7% 85.7% 80.0% 75.0% SEQ ID No. 3 100% 87.5%77.8% 57.1% 80.0% 75.0% SEQ ID No. 4 100% 87.5% 77.8% 57.1% 80.0% 75.0%SEQ ID No. 5 100% 87.5% 77.8% 57.1% 80.0% 75.0% SEQ ID No. 6 100% 75.0%77.8% 85.7% 80.0% 87.5% SEQ ID No. 7 100% 87.5% 66.7% 71.4%  100% 50.0%SEQ ID No. 8 100% 62.5% 44.4% 57.1% 80.0% 62.5% SEQ ID No. 9 100% 12.5%44.4% 42.9% 60.0% 62.5% SEQ ID No. 10 100% 62.5% 55.6% 71.4% 80.0% 50.0%SEQ ID No. 11 100% 50.0% 55.6% 57.1% 80.0% 50.0% SEQ ID No. 12 100%12.5% 22.2% 57.1% 80.0% 52.5% SEQ ID No. 13 100% 50.0% 44.4% 57.1% 80.0%75.0% SEQ ID No. 14  0%  8.3%   0% 14.3%   0% 25.0% SEQ ID No. 15 100%87.5%  100% 85.7%  100% 75.0% Sequence identity relative to the SEQ IDNo. 1 Segment Segment Segment Polypeptide M N O SEQ ID No. 1  100%  100% 100% SEQ ID No. 2 87.5% 80.0% 81.8% SEQ ID No. 3 87.0% 80.0% 81.8% SEQID No. 4 87.5% 80.0% 81.8% SEQ ID No. 5 87.5% 80.0% 81.8% SEQ ID No. 687.5% 80.0% 72.7% SEQ ID No. 7 75.0% 40.0% 36.4% SEQ ID No. 8 75.0%60.0% 54.5% SEQ ID No. 9 62.5% 40.0% 54.5% SEQ ID No. 10 62.5% 40.0%54.5% SEQ ID No. 11 75.0% 40.0% 54.5% SEQ ID No. 12  100% 40.0% 54.5%SEQ ID No. 13 50.0% 40.0% 63.6% SEQ ID No. 14  6.2%   0%   0% SEQ ID No.15 87.5% 80.0% 63.6%

Example 3 Hydrolysis of ZEN by Polypeptides in Cell Lysates

To determine their ability to degrade ZEN into the nontoxic or lesstoxic metabolites HZEN and DHZEN, the polypeptide with the sequence IDno. 1, coded by the nucleotide sequence having the sequence ID no. 17was synthesized as such and with a C-terminal and/or N-terminal 6×Histag in E. coli as described in example 1. The polypeptides with theamino acid sequences having the sequence ID numbers 2 to 15 which werecoded by the nucleotide sequences having the sequence ID numbers 18 to31, were labeled with 6×His exclusively at the C-terminus. 100 mLportions of an E. coli culture having an optical density (OD 600 nm) of2.0-2.5 were harvested by centrifugation at 4° C. and resuspended in 20mL Brunner mineral medium (DSMZ microorganisms medium number 462, 2012).The cell suspensions were lysed by treating three times with a Frenchpress at 20,000 psi. The resulting cell lysates were used in a 1:10,1:100 or 1:1000 dilution prepared in Brunner mineral medium including0.1 mg/mL BSA (bovine serum albumin). For the ZEN degradationexperiments, 9.9 mL Brunner mineral medium was used, including 0.1 mg/mLBSA, 0.1 mL dilute cell lysate and 31 μL ZEN substrate stock solution.On the whole, the cell lysates were thus diluted 1:1000, 1:10,000 and/or1:100,000. The ZEN substrate stock solution used was a 2.08 mM ZENsolution (40 vol % CAN+60 vol % H₂O). To prepare this solution, ZEN incrystalline form (Biopure Standard from Romer Labs, article no. 001109,purity at least 98%) was weighed and dissolved accordingly. Eachdegradation batch was carried out in 25 mL glass vials and incubated at25° C. and 100 rpm for a total of 120 hours with agitation. At the times0, 0.5, 1, 2, 5, 24, 47, 72 and 120 h, a sample of 1 mL was taken eachtime, the polypeptides were heat inactivated for 10 minutes at 99° C.and stored at −20° C. After thawing the sample, the insolubleconstituents were separated by centrifugation. ZEN, HZEN and DHZEN wereanalyzed by means of LC/MS/MS. To do so, the metabolites were separatedchromatographically on a Phenomenex Luna C18(2) column having thedimensions 250 mm×3 mm and a particle size of 5 μm, using as the mobilephase an acetonitrile-water mixture with a formic acid concentration of1 mL/L. The UV signal at 270 nm was recorded using electrosprayionization (ESI) as the ionizing source. ZEN, HZEN and DHZEN werequantified by means of QTrap/LC/MS/MS (triple quadrupole, AppliedBiosystems) in the enhanced mode. After 24 hours at the latest,substantial amounts of ZEN could not be detected any more in any of thebatches. Most of the ZEN, i.e., more than 80%, was converted into HZENor DHZEN.

FIG. 1 shows the degradation of ZEN over time and the increase in HZENas well as DHZEN for a 1:10,000 diluted cell lysate solution as anexample for untagged (FIG. 1A) as well as for C-terminal 6×His tagged(FIG. 1B) and N-terminal 6×His tagged (FIG. 1C) polypeptide with thesequence ID no. 1. It can be seen here clearly that 1) the reaction ofZEN takes place directly and completely because almost no ZEN could bedetected any longer in the first sample (0 h), which was takenimmediately after the start of the experiment, and 2) no mentionablelosses of activity occurred as a result of attaching a tag, whetherC-terminal or N-terminal.

Example 4 Hydrolysis of ZEN Derivatives by Polypeptides in Cell Lysates

To determine the capability of polypeptides to also transform ZENderivatives, in addition to ZEN, into nontoxic and/or less toxicmetabolites, the polypeptides having the sequence ID numbers 1 to 15were prepared as described in Example 3 with C-terminal His tag and therespective synthetic nucleotide sequences with the sequences havingsequence ID numbers 17 to 31 were used as the cell lysates indegradation 15.

The degradation experiments were performed as described in Example 3,where each polypeptide was tested with each ZEN derivative selected fromthe group comprised of α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN,The cell lysates were used in a total dilution of 1:10,000. Instead of a2.08 mM ZEN solution (40 vol % CAN+60 vol % H₂O), equimolar, i.e., 2.08mM solutions of the ZEN derivatives were used as the substrate stocksolution. α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were obtained from Sigmaand used as standards for the analysis. Z14G and Z14S were prepared in apurity of at least 90% according to the methods such as those describedby P. Krenn et al., 2007 (Mykotoxin Research, 23, 4, 180-184) and M.Sulyok at al., 2007 (Anal. Bioanal. Chem. 289, 1505-1523) and used asstandards for the analysis. Another difference in comparison withExample 3 is that only one sample was taken, namely after 24 hours. Thereduction in concentration of the ZEN derivatives during the degradationexperiment was quantified by means of LC/MS/MS. α-ZEL, β-ZEL, Z14G andZ14S were measured by the method of M. Sulyok et al. (2010, FoodChemistry, 119, 408-416); α-ZAL, β-ZAL and ZAN were measured by themethod of P. Songsermaskul et al. (2011, J. of Animal Physiol. andAnimal Nutr., 97, 155-161). It was surprisingly found that only 0 tomax. 13% of the starting amounts of the ZEN derivatives was presentafter 24 hours of incubation in all the degradation experiments.

Example 5 Specific Activity and Enzyme Kinetic Parameters of thePolypeptides as Well as Variants Thereof

The specific activity of the polypeptides and variants thereof wasdetermined photometrically, wherein all the polypeptides used had aC-terminal 6×His tag. The preparation, enrichment and purification ofthe polypeptides and/or variants thereof were performed as described inexample 1. Degradation of ZEN to HZEN was measured on the basis of thereduction in absorption at the wavelength of 315 nm. The molarextinction coefficients (ε) of ZEN and HZEN were determinedexperimentally and were found to amount to 0.0078895 L μmol⁻¹ cm⁻¹ and0.0030857 L μmol⁻¹ cm⁻¹. The extinction coefficients have a strongdependence on pH and therefore the activity must always be measuredprecisely at the same pH and preferably also in the same matrix. Themeasurements were performed in a 50 mM Tris-HCl pH=8.2 buffer solutionin quartz cuvettes in a wavelength range of 200 to 2500 nm in a UV-VISphotometer (Hitachi U-2001) at 32° C.

A 2.08 mM ZEN solution (40 vol % ACN+60 vol % H₂O) was used as the ZENsubstrate stock solution. To prepare this solution, ZEN in crystallineform (Biopure Standard from Romer Labs, article no. 001109, purity atleast 98%) was weighed and dissolved accordingly. The ZEN substratedilutions (0.79 μM, 1.57 μM, 2.36 μM, 3.14 μM, 4.71 μM, 6.28 μM, 7.85μM, 9.42 μM, 10.99 μM, 12.56 μM, 14.13 μM, 15.71 μM, 17.28 μM and 18.85μM) were prepared with 50 mM Tris-HCl pH=8.2. The polypeptide solutionswere diluted to a final concentration of approximately 70 ng/mL using 50mM Tris-HCl buffer pH=8.2. The ZEN substrate dilutions were preheated to32° C. in a water bath.

100 μL portions of the respective ZEN substrate dilution were mixed with0.2 μL polypeptide solution, and the absorption was measured for 5minutes, whereupon each combination of polypeptide solution and ZENsubstrate dilution was measured at least twice.

Taking into account the extinction coefficients of ZEN and HZEN, thereaction rate was calculated for each substance concentration on thebasis of the slope in the absorption over time.

The designations “K_(M) value” or “Michaelis-Menten constant” relate toa parameter for describing the enzymatic affinity of the units μM or mM,which are calculated with the help of the linear Hanes plots accordingto H. Bisswang (2002, Enzyme Kinetics, ISBN 3-527-30343-X, page 19),wherein the function “enzyme kinetics, single substrate” in theSigmaPlot 12.0 program is preferably used for this purpose. Thedesignations “catalytic constant of the enzyme reaction” or “k_(cat)value” relate to a parameter for describing the conversion rate of apolypeptide and/or enzyme, which is given in s⁻¹ and is preferablycalculated with the help of the “enzyme kinetic, single substrate”function of the SigmaPlot 12.0 program. The “maximum enzyme rate” or“v_(max) value” is given in units of μM/s or mM/s and is determined withthe help of the linear Hanes plot by analogy with the K_(M) value,wherein the function “enzyme kinetic, single substrate” of the SigmaPlot12.0 program is preferably used for this.

The specific activity was calculated by means of v_(max) and the enzymeconcentration used according to the equation

${{Specific}\mspace{14mu} {activity}\mspace{14mu} \left( {U\text{/}{mg}} \right)} = \frac{V_{\max}\mspace{14mu} \left( {\mu \; M\text{/}s} \right) \times 60\mspace{14mu} \left( {s\text{/}\min} \right)}{{enzyme}\mspace{14mu} {concentration}\mspace{14mu} \left( {{mg}\text{/}L} \right)}$

wherein one unit is defined as hydrolysis of 1 μmol ZEN per minute at32° C. in 50 mM Tris-HCl buffer solution, pH=8.2.

The raw data for determination of the enzyme parameters K_(M), v_(max),k_(cat) and the specific activity are given below for the polypeptidehaving the sequence ID no. 1. Table 3 shows the reaction rates at therespective ZEN substrate concentrations, while FIG. 2 shows therespective Michaelis-Menton graphs and Table 4 shows the correspondingenzyme kinetic parameters. The enzyme solution that was used had aconcentration of 68 ng/L.

TABLE 3 Reaction rates of the polypeptide with sequence ID no. 1 atdifferent ZEN concentrations. ZEN substrate Measurement 1 Measurement 2dilution reaction rate reaction rate (μM) (μM/s) (μM/s) 0.79 0.00730.0071 1.57 0.0087 0.0082 2.36 0.0095 0.0080 3.14 0.0101 0.0073 4.710.0103 0.0087 6.28 0.0096 0.0088 7.85 0.0084 0.0088 9.42 0.0111 0.008710.99 0.0093 0.0081 12.56 0.0100 0.0086 14.13 0.0089 0.0101 15.71 0.00890.0090 17.28 0.0100 0.0074 18.85 0.0100 0.0085

TABLE 4 Enzyme kinetics parameters of the polypeptide having sequence IDno. 1 Specific activity v_(max) (μM/s) K_(M) (μM) k_(cat) (s⁻¹) (U/mg)Measurement 1 2 1 2 1 2 1 2 Value 0.00993 0.008756 0.2172 0.1898 5.444.79 8.76 7.73 Average 0.009343 0.2035 5.12 8.25

The specific activities of the polypeptides tested are 8.25 U/mg forsequence ID no. 1, 10.56 U/mg for sequence ID no. 2, 8.36 U/mg forsequence ID no. 3, 8.33 U/mg for sequence ID no. 4, 8.56 U/mg forsequence ID no. 5, 9.95 U/mg for sequence ID no. 6, 3.83 U/mg forsequence ID no. 7, 2.57 U/mg for sequence ID no. 8, 4.87 U/mg forsequence ID no. 9, 5.12 U/mg for sequence ID no. 10, 3.88 U/mg forsequence ID no. 11, 2.78 U/mg for sequence ID no. 12, 6.43 U/mg forsequence ID no. 13, 3.33 U/mg for sequence ID no. 14 and 7.76 U/mg forsequence ID no. 15.

The specific activities of the polypeptide variants tested are listed inTable 5 and Table 6.

TABLE 5 Specific activity of functional variants of the polypeptideshaving sequence ID no. 1; conserved amino acid sequence segments, inwhich the mutation(s) is/are located, and sequence identity of thefunctional variants for the parental sequence having sequence ID no. 1.The position of the mutations is given in relation to the amino acidsequence having sequence ID no. 1. The sequence identity was determinedby means of BLAST, as described in Example 2. Muta- Identity Specifiction(s) with SEQ activity n Mutation(s) in range ID No. 1 (U/mg)ZH1-A-001 N25D A 99.7% 8.10 ZH1-A-002 F27Y A 99.7% 7.93 ZH1-A-003 F27H A99.7% 7.78 ZH1-A-004 R35K A 99.7% 8.98 ZH1-A-005 R35Q A 99.7% 8.56ZH1-A-006 N25D/S29P/V42I/V43T A 98.8% 7.84 ZH1-A-007 I26V/R31A/F32Y/F46YA 98.8% 8.61 ZH1-A-S02 N25D/I26V/F27Y/S29P/ A 96.6% 8.73R31A/F32Y/R35K/V37A/ V42I/V43T/F46Y ZH1-A-S03 N25D/I26V/F27H/S29P/ A97.0% 8.52 R31A/F32Y/R35Q/V42I/ V43T/F46Y ZH1-B-001 D53G B 99.7% 8.10ZH1-B-002 N54M B 99.7% 8.41 ZH1-B-003 N54R B 99.7% 8.33 ZH1-B-004 S69G B99.7% 8.06 ZH1-B-005 P72E B 99.7% 8.65 ZH1-B-006 P72R B 99.7% 8.78ZH1-B-S02 N54M/L57V/L60I/S69G/ B 98.2% 8.51 P72E/V73A ZH1-B-S03D53G/N54R/L57V/L60I/ B 98.2% 8.56 P72E/V73A ZH1-B-S04N54R/L57V/L60I/P72E/ B 98.5% 8.96 V73A ZH1-B-S14 N54R/L58V/L59P/L60V/ B97.6% 8.68 T64G/P72R/G75P/L77P ZH1-C-001 N80H C 99.7% 8.24 ZH1-C-002N80D C 99.7% 8.48 ZH1-C-003 F84Y C 99.7% 8.65 ZH1-C-S06 N80H/F84Y C99.4% 8.88 ZH1-C-S10 N80H/F84H C 99.4% 8.32 ZH1-C-S14 E79R/N80D C 99.4%8.45 ZH1-D-001 T95S D 99.7% 8.53 ZH1-D-002 R99K D 99.7% 8.25 ZH1-D-003V123I D 99.7% 8.17 ZH1-D-004 A125G D 99.7% 8.36 ZH1-D-005 G126A D 99.7%8.41 ZH1-D-006 G130A D 99.7% 8.69 ZH1-D-007 G130V D 99.7% 8.54 ZH1-D-008G131A D 99.7% 8.71 ZH1-D-009 N127D D 99.7% 8.29 ZH1-D-010 N127Q D 99.7%8.34 ZH1-D-011 A141S D 99.7% 8.67 ZH1-D-012 F106W D 99.7% 7.84 ZH1-D-013I118V D 99.7% 8.37 ZH1-D-014 I118V/V123L D 99.4% 8.55 ZH1-D-015I118V/K119R/L132V D 99.1% 8.86 ZH1-D-016 W96Q/F106W/L116G/ D 98.8% 8.65V122A ZH1-D-017 Q91R/N105D/K119G/ D 98.5% 8.46 A141S/M142K ZH1-D-S02T95S/T97A/R99K/ D 97.7% 8.66 I118V/V123I/L132V/ A141S ZH1-D-S03T95S/R99K/I118V/ D 98.2% 9.32 K119R/L132V/A141S ZH1-D-S04T95S/R99K/I118V/ D 98.5% 9.15 L132V/A141S ZH1-D-S05 T95S/R99K/I114M/ D97.7% 8.84 I118V/K119R/L132V/ A141S ZH1-D-S07 R99G/A115D/K119G/ D 96.3%8.79 P121T/V123I/A125S/ L132V/L133V/S138A/ Y140F/A141S/M142L ZH1-D-S08R93K/W96Q/R99G/ D 97.0% 8.86 D104N/N105L/F106M/ A115S/V123I/A125S/ G144NZH1-D-S09 R99G/S102N/D104N/ D 95.4% 8.99 N105T/F106W/L110V/V111E/A115D/K119G/ V122T/V123L/L132V/ L133I/S138A/M142K ZH1-D-S10W96R/S102T/F106I/ D 96.0% 9.12 I114L/A115S/L116G/ K119G/V122A/V123F/A125S/A134S/Y140F/ M142E ZH1-D-S11 W96R/R99G/S102T/ D 95.1% 8.54F106V/I114L/A115D/ L116G/K119G/V122A/ V123F/A125S/N127L/L133A/A134S/Y140F/ M142K ZH1-D-S12 S94T/R99G/S102T/ D 95.1% 8.69N105I/L110V/A115D/ K119G/P121E/V122T/ V123L/V124I/L133I/A134G/S138A/Y140F/ M142K ZH1-D-S13 R93Q/R99G/N105T/ D 96.0% 8.47R112K/A115D/L116I/ A125S/N127L/L132V/ L133V/A134S/Y140F/ M142K ZH1-D-S14Q91R/W96R/N105D/ D 97.3% 8.55 I114L/I118V/K119R/ V122A/L132V/L137SZH1-E-001 Y165C E 99.7% 8.46 ZH1-E-002 Y165H E 99.7% 8.33 ZH1-E-003P163T E 99.7% 7.95 ZH1-E-004 A154P/Y165C E 99.4% 8.13 ZH1-E-S02P163T/A164T/Y165C/ E 98.5% 8.83 V169I/L170R ZH1-E-S05 A154P/Y165H/L170RE 99.1% 9.65 ZH1-F-001 Y180F F 99.7% 8.35 ZH1-F-002 D182T F 99.7% 8.41ZH1-F-003 D182K F 99.7% 8.19 ZH1-F-004 Y180F/R181V/I190V F 99.1% 8.56ZH1-F-S04 Y180F/D182T/F183Y/ F 98.5% 8.56 I190V/G191S ZH1-F-S06Y180F/D182T/F183Y/ F 98.8% 8.64 I190V ZH1-F-S10 E178A/R181V/D182K/ F98.8% 7.55 F183Y ZH1-H-001 T236K H 99.7% 8.09 ZH1-H-002 V237F H 99.7%8.11 ZH1-H-003 E234G H 99.7% 8.54 ZH1-H-S02 F233W H 99.7% 8.37 ZH1-H-S03F233Y H 99.7% 8.64 ZH1-H-S04 F233H H 99.7% 8.36 ZH1-H-S06 A231V/F233Y H99.4% 8.54 ZH1-H-S09 F232W/F233A/E234T/ H 98.5% 8.83 G235D/L239AZH1-I-001 H240N I 99.7% 8.54 ZH1-I-002 H240S I 99.7% 8.79 ZH1-I-003D244E/R245Y I 99.4% 8.42 ZH1-I-S02 D244E/R245Q/M246L I 99.1% 8.36ZH1-I-S03 H240N/D244E I 99.4% 9.26 ZH1-I-S06 H240S/D244E I 99.4% 9.02ZH1-I-S07 L239Q/H240T/R245Y I 99.1% 8.41 ZH1-J-001 Q249R J 99.7% 8.36ZH1-J-002 T252V J 99.7% 7.94 ZH1-J-S02 I254V J 99.7% 8.55 ZH1-J-S03Q249R/K251N/I254V J 99.1% 9.03 ZH1-J-S07 T252V/I254M J 99.4% 7.81ZH1-J-S10 T252V/I254V J 99.4% 7.97 ZH1-K-S05 A260M K 99.7% 8.64ZH1-K-S11 A260F K 99.7% 8.82 ZH1-K-S13 A260S K 99.7% 9.01 ZH1-L-001E266Y L 99.7% 8.46 ZH1-L-002 E266D L 99.7% 8.31 ZH1-L-003 T262G L 99.7%8.32 ZH1-L-004 T262D/E266D/ L 99.4% 8.56 ZH1-L-005 T262G/I283T/ L 99.4%8.68 ZH1-L-S02 E266D/E269H L 99.4% 8.59 ZH1-L-S04 I263T/E269N L 99.4%8.73 ZH1-L-S06 E269N L 99.7% 8.69 ZH1-L-S13 E266Y/E269N L 99.4% 8.33ZH1-M-001 L274M M 99.7% 8.29 ZH1-M-002 L274C M 99.7% 8.37 ZH1-M-S02L277E M 99.7% 8.96 ZH1-M-S07 L274M/A279V M 99.4% 8.23 ZH1-M-S08L274T/L277F M 99.4% 8.63 ZH1-M-S11 L274C/L277I M 99.4% 8.51 ZH1-N-001H297L N 99.7% 8.27 ZH1-N-002 H298V/L302S N 99.4% 9.03 ZH1-N-S02 H298V N99.7% 8.94 ZH1-N-S09 H298L/P299D N 99.4% 8.37 ZH1-O-001 L307Q O 99.7%8.62 ZH1-O-002 F308S O 99.7% 8.57 ZH1-O-S02 L307Q/A311P O 99.4% 8.34ZH1-O-S03 L307Q/F308S O 99.4% 8.74 ZH1-O-S06 L307Q/F308S/D309A O 99.1%9.18 ZH1-B/ D53G/N54R/L57V/ B + H 97.3% 9.26 H-001 L60I/P72E/V73A/F233V/E234G/V237F ZH1-C/ N80H/F84Y/T95S/ C + D 97.6% 9.31 D-001R99K/I118V/K119R/ L132V/A141S ZH1-D/ T95S/T97A/R99K/ D + K 97.6% 9.66K-001 I118V/V123I/L132V/ A141S/A260M ZH1-D/ T95S/T97A/R99K/ D + M 97.6%10.63 M-001 I118V/V123I/L132V/ A141S/L277E ZH1-K/ A260M/H298V K + N99.4% 8.94 N-001 ZH1-K/ A260M/T262D/E266D/ K + L 98.8% 9.03 L-001 E269HZH1-K/ A260M/T262G/I263T/ K + L 98.8% 8.84 L-002 E269N ZH1-N/Q296A/H298V/L307Q/ N + O 98.8% 9.26 O-001 A311P ZH1-N/Q296E/H298V/L302S/ N + O 98.5% 9.46 O-002 L307Q/F308S ZH1-C/D/N80H/F84Y/T95S/ C + D + 97.0% 9.97 J-001 R99K/I118V/L132V/ JA141S/Q249R/K251N/ I254V ZH1-B/D/ D53G/N54R/L57V/L60I/ B + D + 95.7%10.78 K-001 P72E/V73A/T95S/R99K/ K I114M/I118V/K119R/ L132V/A141S/A260MZH-J/K/ I254V/I256L/A260M/ J + K + 98.2% 9.11 L-001 T262G/I263T/E269N LZH1-J/K/ I254V/I256L/A260M/ J + K + 97.7% 9.14 LM-001 T262D/E266D/E269H/L + M L271V ZH1-B/C/D/ E79R/N80D/D53G/ B + C + 92.1% 11.31 J-002N54R/L57V/L60I/ D + J P72E/V73A/W96R/ R99G/S102T/F106V/I114L/A115D/L116G/ K119G/V122A/V123F/ A125S/N127L/L133A/A134S/Y140F/M142K/ T252V/I254V ZH1-DEL- ΔP212 — 99.7% 8.56 001 ZH1-DEL-ΔG5/ΔT6/ΔR7/ — 95.4% 8.37 002 ΔS8/ΔE9/ΔA10/ ΔA11/ΔD12/ΔA13/ΔA14/ΔT15/ΔQ16/ ΔA17/ΔR18/ΔQ19 ZH1-DEL- ΔN327/ΔD328 — 99.4% 8.27 003ZH1-A/B/ N25D/I26V/F27Y/ A + B + 89.6% 9.54 C-001 S29P/R31A/F32Y/ CR35K/V37A/V42I/ V43T/F46Y/N54R/ L58V/L59P/L60V/ T64G/P72R/G75P/L77P/R99G/S102N/ D104N/N105T/F106W/ L110V/V111E/A115D/K119G/V122T/V123L/ L132V/L133I/S138A/ M142K ZH1-DEL/ ΔG5/ΔT6/ΔR7/ B +C + 86.6% 11.52 B/C/D/ ΔS8/ΔE9/ΔA10/ D + J J-001 ΔA11/ΔD12/ΔA13/ΔA14/ΔT15/ΔQ16/ ΔA17/ΔR18/ΔQ19/ ΔP212/ΔN327/ΔD328/ E79R/N80D/D53G/N54R/L57V/L60I/ P72E/V73A/W96R/ R99G/S102T/F106V/ I114L/A115D/L116G/K119G/V122A/V123F/ A125S/N127L/L133A/ A134S/Y140F/M142K/ T252V/I254VZH1-DEL/ ΔG5/ΔT6/ΔR7/ A + B + 83.3% 10.92 A/B/C/ ΔS8/ΔE9/ΔA10/ C + D +D/J-001 ΔA11/ΔD12/ΔA13/ J ΔA14/ΔT15/ΔQ16/ ΔA17/ΔR18/ΔQ19/ΔP212/ΔN327/ΔD328/ N25D/I26V/F27Y/ S29P/R31A/F32Y/ R35K/V37A/V42I/V43T/F46Y/E79R/ N80D/D53G/N54R/ L57V/L60I/P72E/ V73A/W96R/R99G/S102T/F106V/I114L/ A115D/L116G/K119G/ V122A/V123F/A125S/N127L/L133A/A134S/ Y140F/M142K/T252V/ I254V ZH1-001 L302S — 99.7% 8.31

TABLE 6 Specific activities of functional variants of the polypeptidehaving sequence ID no. 2. The position of the mutation(s) is relative tothe amino acid sequence with sequence ID no. 2. The sequence identitywas determined by means of BLAST as described in example 2. IdentitySpecific with SEQ activity Variant Mutation(s) ID No. 2 (U/mg) ZH2-001D3D(GTRSEAADAATQARQL) 93.6% 10.15 ZH2-002 D8N/V9I/Y10F 99.0% 10.42ZH2-003 M37N/E55P/A56V/ 97.0% 10.58 V101I/S124A/F194FP/T146P/T147A/C148Y ZH2-004 S187P/S188A/P189K/ 97.7% 10.43M190A/A191M/R192Q/ Y193L ZH2-005 A262E/R263H/R265Q/ 97.7% 10.68L266D/L267I/M268I/ E269R ZH2-006 D3D(GTRSEAADAATQARQL)/ 86.1% 10.71M37N/E55P/A56V/ V101I/S124A/F194FP/ T146P/T147A/C148Y/S187P/S188A/P189K/ M190A/A191M/R192Q/ Y193L/A262E/R263H/R265Q/L266D/L267I/ M268I/E269R

Example 6 Degradation of ZEN and ZEN Derivatives in Contaminated Corn

To determine the capabilities of polypeptides to degrade naturallyoccurring ZEN and ZEN derivatives in a complex matrix and at a low pH,contaminated corn was mixed with different concentrations of one of thepolypeptides having the sequence ID numbers 1 to 6 and the degradationof ZEN and ZEN derivatives was tracked.

The contaminated corn was ground and used in the degradation experimentwherein a batch would consist of 1 g ground contaminated corn, 8.9 mL100 mM acetate buffer pH 4.0 and 0.1 mL polypeptide solution. Enrichedand purified polypeptide solutions were prepared as described in example5, diluting them to a concentration of 10 mU/mL, 100 mU/mL and/or 1000mU/mL. Thus in absolute amounts 1 mU (=1 mU per gram corn), 10 mU (=10mU per gram corn) and/or 100 mU (=100 mU per gram of corn) were used inthe batch. Each degradation batch was carried out in 25 mL and incubatedat 37° C. and 100 rpm with agitation. Before adding the enzyme and/orafter 1 hour of incubation, a sample of 1 mL was taken, the polypeptidewas heat inactivated at 99° C. for 10 minutes and the sample was storedat −20° C. After thawing the sample, the insoluble constituents wereseparated by centrifugation. Concentrations of ZEN and ZEN derivativeswere measured by means of LC/MS/MS as described by M. Sulyok et al.(2007, Anal. Bioanal. Chem., 289, 1505-1523). The ZEN and ZEN derivativecontent in this corn was 238 ppb for ZEN, 15 ppb for α-ZEL, 23 ppb forβ-ZEL, 32 ppb for Z14G and 81 ppb for Z14S. Table 7 shows the percentagereduction in the ZEN and ZEN derivative content in the degradationexperiment.

TABLE 7 Reduction in ZEN and ZEN derivatives in percentage based on thestarting content in the degradation experiment with differentpolypeptides and amounts of polypeptides. Amount in Polypeptide thebatch ZEN α-ZEL β-ZEL Z14G Z14S SEQ 0.1 mU 83% ≧80% 70%  78% 80% ID No.1 1 mU 96% ≧80% 76% ≧80% 92% 10 mU 97% ≧80% ≧85%  ≧80% 94% SEQ 0.1 mU87% ≧80% 73% ≧80% 84% ID No. 2 1 mU 97% ≧80% 78% ≧80% 90% 10 mU 99% ≧80%≧85%  ≧80% 96% SEQ 0.1 mU 79%  79% 67%  73% 75% ID No. 3 1 mU 85% ≧80%72%  79% 82% 10 mU 92% ≧80% 78% ≧80% 88% SEQ 0.1 mU 82%  78% 65%  76%80% ID No. 4 1 mU 89% ≧80% 73% ≧80% 86% 10 mU 93% ≧80% 82% ≧80% 91% SEQ0.1 mU 79%  76% 66%  78% 80% ID No. 5 1 mU 83% ≧80% 73% ≧80% 81% 10 mU91% ≧80% 79% ≧80% 86% SEQ 0.1 mU 93% ≧80% 75% ≧80% 90% ID No. 6 1 mU 95%≧80% 82% ≧80% 92% 10 mU 98% ≧80% ≧85%  ≧80% 96%

Example 7 Additives Containing Polypeptide for Hydrolytic Cleavage ofZEN and/or ZEN Derivatives

To prepare additives for hydrolytic cleavage of ZEN, fermentationsupernatants of polypeptides expressed by P. pastoris and having thesequence ID numbers 1, 2, 6 and 13 were purified by microfilitration andultrafiltration (exclusion limit: 10 kDa) under standard conditions andconcentrated up to a dry substance concentration of approximately 9% byweight. Following that, these polypeptide-containing solutions were alsoprocessed further to form dry powders under standard conditions in aspray dryer (Mini B290 from Büchi). These four powders were subsequentlydesignated as Z1, Z2, Z6 and Z13. Z1, Z2, Z6 and/or Z13 wereadditionally mixed with bentonite having an average grain size ofapproximately 1 μm in a ratio of 1% by weight of additives Z1, Z2, Z6and/or Z13 and 99% by weight bentonite in an overhead agitator. Theresulting additives are designated as additives Z1.B, Z2.B, Z6.B andZ13.B. In addition, Z1, Z2, Z6 and Z13 were mixed with bentonite and avitamin trace element concentrate in a ratio of 0.1% by weight additiveZ1, Z2, Z6 and/or Z13, 0.9% by weight vitamin trace elements concentrateand 99% by weight bentonite in an overhead agitator. The resultingadditives were designated as additive Z1.BVS, Z2.BVS, Z6.BVS andZ13.BVS. 100 g of the additives Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVScontained 200 mg iron sulfate, 50 mg copper sulfate, 130 mg zinc oxide,130 mg manganese oxide, 2.55 mg calcium carbonate, 160 mg vitamin E, 6.5mg vitamin K3, 6.5 mg vitamin B1, 14 mg vitamin B2, 15 mg vitamin B6,0.15 mg vitamin B12, 150 mg nicotinic acid, 30 mg pantothenic acid and5.3 mg folic acid.

The additives were extracted for 30 minutes in a 50 mM Tris-HCl bufferpH=8.2 and diluted further in the same buffer so that the finalconcentration of polypeptide was approximately 70 ng/mL.

Following that, the zearalenone-degrading effect of these solutions wasdetermined as described in Example 5. The corresponding activities were8.230 U/g for Z1, 9.310 U/g for Z2, 9.214 U/g for Z6, 83 U/g for Z1.B,92 U/g for Z2.B, 90 U/g for Z2.C, 57 U/g for Z13.B, 8 U/g for Z1.BVS, 9U/g for Z2.BVS, 9 U/g for Z6.BVS and 6 U/g for Z13.BVS.

The ability to degrade ZEN derivatives α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G,Z14S and ZAN by the additives Z1, Z2, Z6, Z13, Z1.B, Z2.B, Z6.B, Z13.B,Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS was tested as described in Example 4,but instead of 100 μL of a cell lysate, 100 μL of a polypeptide solutionwith a polypeptide concentration of approximately 70 ng/mL was used.After incubating for 6 hours, only max. 15% of the starting amount waspresent as unhydrolyzed ZEN derivative.

Example 8 Optimum Temperature

To determine the temperature optimum of the polypeptides having SEQ IDnumbers 1, 2, 5, 6, 7, 9, 11, 12 and 15, they were cloned with aC-terminal 6×His tag as described in example 1, expressed in E. coli andpurified. In preliminary experiments, the concentration at which acomplete conversion of ZEN could be ensured under the experimentalconditions was determined (Teorell-Stenhagen buffer (Teorell andStenhagen, A universal buffer for the pH range of 2.0 to 12.0. BiochemZtschrft, 1938, 299:416-419), pH 7.5 with 0.1 mg/mL BSA at 30° C.) afteran experimental time of 3 hours. The preparations were used in theconcentrations thus determined in the degradation batches fordetermining the optimum temperature. The experiments were carried out ina PCR Cycler (Eppendorf) using the temperature gradient function at 20°C.±10° C., at 40° C.±10° C. and, if necessary, at 60° C.±10° C. (10temperatures in the respective range; temperatures predefined by the PCRcycler). For the batches Teorell-Stenhagen buffer was mixed with thecorresponding enzyme concentration and 0.1 mg/mL BSA plus 5 ppm ZEN atthe respective optimum pH. Batches with 0.1 mg/mL BSA and 5 ppm ZENwithout addition of an enzyme were used as negative controls. After 0 h,0.5 h, 1 h, 2 h and 3 h incubation time, a sample was taken perincubation temperature, heat inactivated for 10 minutes at 99° C. andstored at −20° C. After thawing, the samples were transferred to HPLCvials. ZEN, HZEN and DHZEN were analyzed by HPLC-DAD. To do so themetabolites were separated chromatographically on a Zorbax SB-Aq C18column with the dimensions 4.6 mm×150 mm and a particle size of 5 μm. Amethanol-water mixture with 5 mM ammonium acetate was used as the mobilephase. The UV signal at 274 nm was recorded. The metabolites werequantified by including entrained standard series. The optimumtemperatures were determined on the basis of the slopes determined forthe degradation curves, where the optimum temperature was defined as thetemperature at which the slope was the greatest. Table 8 shows theoptimum temperatures.

TABLE 8 Optimum temperatures of the polypeptides. SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 1 No. 2 No. 5 No. 6 No. 7No. 9 No. 11 No. 12 No. 15 38° C. 41° C. 50° C. 51° C. 31° C. 35° C. 50°C. 26° C. 41° C.

Example 9 Thermal Stability

To determine the thermal stability of polypeptides with the SEQ IDnumbers 1, 2, 5, 6, 7, 9, 11, 12 and 15, they were cloned with aC-terminal 6×His tag as described in Example 1, expressed in E. coli andpurified. They were then incubated in the PCR cycler with a gradientfunction at the respective optimum temperature ±10° C. After 0 min, 15min, 30 min and 60 min, one sample was taken per batch and pertemperature. These pre-incubated samples were then used in a degradationexperiment in the Teorell-Stenhagen buffer at the respective optimum pHwith 0.1 mg/mL BSA and 5 ppm ZEN. In preliminary experiments, theconcentration at which a complete reaction of ZEN could be ensured afteran experimental duration of 3 hours under the experimental conditions(Teorell-Stenhagen buffer, pH 7.5 with 0.1 mg/mL BSA at 30° C.) wasdetermined for each polypeptide. The respective enzyme concentrationthereby determined was used in the batches. The degradation batches wereincubated at 30° C. Sampling was performed after 0 h, 0.5 h, 1 h, 2 hand 3 h Incubation time. Next, the polypeptides were heat-inactivatedfor 10 minutes at 99° C. and the samples were stored at −20° C. Afterthawing the samples were transferred to HPLC vials and analyzed byHPLC-DAD, as described in Example 8.

Thermal stability is defined as the temperature at which thepolypeptides have a 50% residual activity in comparison with the optimumtemperature after 15 minutes of pre-incubation. As a measure of theactivity, the slope in the degradation curves is used. The temperaturestabilities are shown in Table 9.

TABLE 9 Temperature stability of the polypeptides (50% residual activityafter pre-incubation for 15 minutes). SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID No. 1 No. 2 No. 5 No. 6 No. 7 No. 9 No. 11No. 12 No. 15 38° C. 34° C. 54° C. 61° C. 28° C. 44° C. 55° C. 40° C.49° C.

Example 10 Optimum pH

To determine the optimum pH of the polypeptides having the SEQ IDnumbers 1, 2, 5, 6, 7, 9, 11, 12 and 15, they were cloned with aC-terminal 6×His tag as described in Example 1, expressed in E. coli andpurified. In preliminary experiments, the concentration at which acomplete conversion of ZEN could be ensured after an experimentalduration of 3 hours under the experimental conditions was determined foreach polypeptide (Teorell-Stenhagen buffer, pH 7.5 with 0.1 mg/mL BSA at30° C.). The respective enzyme concentration was used in the batches.The degradation batches were carried out in Stenhagen buffer at pHlevels of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 11.0 and 12.0. For the degradation batches with 0.1 mg/mL BSA and5 ppm ZEN, incubation was done at 30° C. Batches in Teorell-Stenhagenbuffer were used as the negative controls at pH 3.0, pH 7.0 and pH 12.0with 0.1 mg/mL BSA and 5 ppm ZEN. Sampling was performed after anincubation time of 0 h, 0.5 h, 1 h, 2 h and 3 h. Next the polypeptideswere heat-inactivated for 10 minutes at 99° C. and the samples werestored at −20° C. After thawing, the samples were transferred to HPLCvials and analyzed by HPLC-DAD as described in Example 8. The optimum pHwas determined on the basis of the slopes found for the degradationcurves, wherein the pH at which the slope was the greatest was definedas the optimum pH. Table 10 shows the optimum pH levels.

TABLE 10 Optimum pH of the polypeptides. SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID No. 1 No. 2 No. 5 No. 6 No. 7 No. 9 No.11 No. 12 No. 15 8.2 8.5 7.0-8.0 7.0-7.5 7.5-8.5 7.0-7.5 8.0 7.0-7.5 7.5

Example 11 pH Stability at pH 5.0

To determine the pH stability, the polypeptides from Example 10 wereincubated for one hour at 25° C. in Teorell-Stenhagen buffer at pH 5.0and at the respective optimum pH. These pre-incubated samples were usedin a degradation experiment in the same concentrations of the respectivepolypeptide as those used to determine the optimum pH in 100 mM Tris-HClbuffer at the respective optimum pH with 0.1 mg/mL BSA and 5 μm ZEN inthe batch. The batches were incubated at the respective optimumtemperature. Sampling was performed after 0 h, 0.5 h, 1 h, 2 h and 3 hincubation time. Next the polypeptides were heat inactivated for 10minutes at 99° C. and the samples were stored at −20° C. After thawing,the samples were transferred to HPLC vials and analyzed by means ofHPLC-DAD as described in Example 8. The pH stability is defined as thepercentage residual activity of the polypeptides at pH 5.0 relative tothe activity at the respective optimum pH. The pH stabilities for 5.0are shown in Table 11.

TABLE 11 pH stability of the polypeptides at pH 5.0. SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 1 No. 2 No. 5 No. 6 No.7 No. 9 No. 11 No. 12 No. 15 3% 17% 79% 80% 100% 22% 87% 98% 19%

Example 12 ZEN Degradation Experiment

The degradation of ZEN to HZEN and DHZEN was performed as an example forthe polypeptides with sequence ID numbers 1, 2, 5, 6, 7, 9, 11, 12 and15. The degradation batches were carried in Teorell-Stenhagen buffer pH7.5 with 0.1 mg/mL BSA and 5 ppm ZEN. The degradation batches wereincubated at 30° C. Sampling was performed after 0 h, 0.5 h, 1 h, 2 hand 3 h incubation time. Next the polypeptides were heat-inactivated for10 minutes at 99° C. and the samples were stored at −20° C. Afterthawing, the samples were transferred to HPLC vials and analyzed byHPLC-DAD, as described in Example 8. The polypeptide concentration wasselected so that complete degradation was achieved after approximately 3hours. FIG. 3 shows the degradation kinetics, where the y axis shows theconcentration of ZEN, HZEN and DHZEN in micromoles per liter (μmol/L)and the x axis shows the incubation time in hours (h).

*μM denotes micromolar and corresponds to the unit μmol/L

1-22. (canceled)
 23. A polypeptide that hydrolytically cleaveszearalenone and/or at least one zearalenone derivative, wherein thepolypeptide is a hydrolase with an amino acid sequence selected from thegroup of sequence ID numbers 1 to 15 or a functional derivative thereof,wherein a sequence identity of at least 70% exists between thefunctional variants and at least one of the amino acid sequences. 24.The polypeptide according to claim 23, wherein the polypeptide has atleast one conserved amino acid sequence segment or a functional variantthereof, wherein the functional variant of the amino acid sequencesegment has a sequence identity of at least 70%, preferably at least84%, more preferably at least 92% and most preferably at least 98%, andat least one conserved amino acid sequence segment is selected from thegroup of amino acid sequences +24 to +50, +52 to +77 +79 to +87, +89 to+145, +150 to +171, +177 to +193, +223 to +228, +230 to +237, +239 to+247, +249 to +255, +257 to +261, +263 to +270, +272 to +279, +297 to+301, +303 to +313, +24 to 328, +1 to +328 of the sequence having thesequence ID no.
 1. 25. The polypeptide according to claim 23, whereinthe polypeptide has at least one mutation of the amino acid sequencewith respect to sequence ID no. 1 in at least one of the followingpositions selected from the group: 22, 23, 25, 26, 27, 29, 31, 32, 35,37, 42, 43, 46, 51, 53, 54, 57, 60, 69, 72, 73, 78, 80, 84, 88, 95, 97,99, 114, 118, 119, 123, 132, 141, 146, 148, 149, 154, 163, 164, 165,169, 170, 172, 176, 180, 182, 183, 190, 191, 194, 196, 197, 198, 201,204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 216, 217, 220, 221,222, 229, 231, 233, 238, 240, 244, 245, 246, 248, 249, 251, 254, 256,260, 262, 263, 266, 269, 271, 277, 280, 281, 282, 283, 284, 285, 286,287, 292, 296, 298, 302, 307, 308, 309, 311, 314, 317, 319, 321, 323,325 and
 326. 26. The polypeptide according to claim 23, wherein it hasat least one mutation of the amino acid sequence with respect tosequence ID no. 1 selected from the group D22A, S23Q, S23L, N25D, I26V,F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V37A, V42I, V43T, F46Y, S51E,S51D, D53G, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S, N80H, F84Y,I88L, T95S, T97A, R99K, I114M, I118V, K119R, V123I, L132V, A141S, I146V,I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R,A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, G191S, K194T, K194E,F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S,K206P, A207M, M208A, Q209R, L210A, L210S, ΔP212, T213V, P214A, E216T,E216G, A217I, N220H, L221M, K222R, K222Q, G229A, A231V, F233W, F233Y,F233H, A238G, H240N, H240S, D244E, R245Q, M246L, S248T, S248N, S248G,Q249R, K251N, I254V, I256L, A260M, T262D, T262G, I263T, E266D, E269H,E269N, L271V, L277E, E280A, E280L, H281R, H281Q, A282V, Q283R, D284L,D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A, Q296E, H298V,L302S, L307Q, F308S, D309A, A311P, A314V, L317F, S319Q, S319P, S319R,S321A, S321T, T323A, P325A, A326P.
 27. The polypeptide according toclaim 23, wherein it contains at least one of the following amino acidmotifs selected from the group of sequence ID numbers 32 to
 69. 28. Theisolated polynucleotide having a nucleotide sequence that codes for apolypeptide, wherein the polypeptide has the property of hydrolyzingzearalenone and/or at least one zearalenone derivative, wherein thenucleotide sequence codes from at least one polypeptide according toclaim 23 and/or the nucleotide sequence has a degree of sequenceidentity of at least 70% for a nucleotide sequence selected from thegroup having sequence ID numbers 16 to 31 and/or the nucleotide sequencehydrolyzes under moderate stringency conditions with at least onenucleotide sequence, selected from the group of sequence ID numbers 16to 31 and/or with a subsequence thereof having at least 200 nucleotides,in particular at least 100 nucleotides and/or a complementary strand ofthe nucleotide sequence or the subsequence thereof.
 29. An additive thathydrolytically cleaves zearalenone and/or at least one zearalenonederivative to yield feed products for pigs, poultry or aquaculture, foraddition to foodstuffs or to distillers dried grain and solubles,wherein the additive contains at least one polypeptide having an aminoacid sequence selected from the group of sequence ID numbers 1 to 15 ora functional variant thereof, wherein a sequence identity between thefunctional variant and at least one of the amino acid sequences amountsto at least 70% and auxiliary substances are also present.
 30. Theadditive that hydrolytically cleaves zearalenone and/or at least onezearalenone derivative to yield feed products for pigs, poultry oraquaculture, for addition to foodstuffs or to distillers dried grain andsolubles, wherein the additive contains at least one polypeptide havingan amino acid sequence selected from the group of sequence ID numbers 1to 15 or a functional variant thereof, wherein a sequence identitybetween the functional variant and at least one of the amino acidsequences amounts to at least 70% and auxiliary substances are alsopresent, wherein at least one polypeptide according to claim 23 iscontained therein.
 31. The additive according to claim 29, wherein theauxiliary substances are selected from at least one inert carrier aswell as optional additional ingredients, such as vitamins and/orminerals and/or enzymes and/or other components for detoxifyingmycotoxins.
 32. The additive that hydrolytically cleaves zearalenoneand/or at least one zearalenone derivative to yield feed products forpigs, poultry or aquaculture, for addition to foodstuffs or todistillers dried grain and solubles, wherein the additive contains atleast one polypeptide having an amino acid sequence selected from thegroup of sequence ID numbers 1 to 15 or a functional variant thereof,wherein a sequence identity between the functional variant and at leastone of the amino acid sequences amounts to at least 70% and auxiliarysubstances are also present, wherein at least one polypeptide accordingto claim 22 is present in the additive in a concentration of at most10,000 U/g, preferably at most 1000 U/g, more preferably at most 100 U/gand most preferably at most 10 U/g.
 33. The additive according to claim29, wherein the additive is present in encapsulated or coated form. 34.A use of at least one polypeptide having an amino acid sequence selectedfrom the group of sequence ID numbers 1 to 15 or a functional variantthereof, wherein additives containing a sequence identity between thefunctional variant and at least one of the amino acid sequences amountsto at least 70%, for hydrolytic cleavage of zearalenone and/or at leastone zearalenone derivative in animal feed products, in particular pigs,poultry and aquaculture, in food items or in distillers dried grain andsolubles.
 35. A method for hydrolytic cleavage of zearalenone and/or atleast one zearalenone derivative, wherein the zearalenone and/or atleast one zearalenone derivative is/are hydrolyzed by a polypeptidehaving an amino acid sequence selected from the group of sequence IDnumbers 1 to 15 or a functional variant thereof, wherein the sequenceidentity between the functional variant and at least one of the aminoacid sequences is at least 70%.
 36. The method for hydrolytic cleavageof zearalenone and/or at least one zearalenone derivative, wherein thezearalenone and/or at least one zearalenone derivative is/are hydrolyzedby a polypeptide having an amino acid sequence selected from the groupof sequence ID numbers 1 to 15 or a functional variant thereof, whereinthe sequence identity between the functional variant and at least one ofthe amino acid sequences is at least 70%, wherein the polypeptide isused in an additive according to claim
 28. 37. The method according toclaim 36, wherein the polypeptide or the additive is mixed with afoodstuff or animal feed product contaminated with zearalenone and/orwith at least one zearalenone derivative; the contaminated foodstuff oranimal feed product is brought in contact with moisture, and thepolypeptide or the additive hydrolyzes the zearalenone and/or at leastone zearalenone derivative present in contaminated foodstuff or animalfeed product.
 38. The method according to claim 35, wherein at least70%, preferably at least 80%, in particular at least 90% of thezearalenone and/or at least one zearalenone derivative is/arehydrolyzed.